Generation of isogenic single and multiplex gene knockout mice by base editing-induced STOP

Guang Yang; Tianyu Zhu; Zongyang Lu; Guanglei Li; Hao Zhang; Songjie Feng; Yajing Liu; Jianan Li; Yu Zhang; Jia Chen; Xuejiang Guo; Xingxu Huang

doi:10.1016/j.scib.2018.07.002

Generation of isogenic single and multiplex gene knockout mice by base editing-induced STOP

Sci Bull (Beijing). 2018 Sep 15;63(17):1101-1107. doi: 10.1016/j.scib.2018.07.002. Epub 2018 Jul 5.

Authors

Guang Yang¹, Tianyu Zhu², Zongyang Lu¹, Guanglei Li¹, Hao Zhang², Songjie Feng¹, Yajing Liu¹, Jianan Li¹, Yu Zhang¹, Jia Chen¹, Xuejiang Guo³, Xingxu Huang⁴

Affiliations

¹ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China.
² State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 211166, China.
³ State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 211166, China. Electronic address: guo_xuejiang@njmu.edu.cn.
⁴ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. Electronic address: huangxx@shanghaitech.edu.cn.

PMID: 36658989
DOI: 10.1016/j.scib.2018.07.002

Abstract

Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain: the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes. Therefore, it takes a long time to get homozygous mutants. Recently developed base editing (BE) system, which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons (i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sgRNA targeting CD160 and a mixture of sgRNAs targeting VISTA and CD160, respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.

Keywords: Base editing; CD160; Isogenic; Knockout; VISTA; i-STOP.